Conclusion

Malgré ces inconvénients, le modèle d’Ussing est un modèle particulièrement pertinent lors du développement préclinique, particulièrement lorsqu’il s’agit de mieux comprendre les mécanismes d’absorptions et / ou de toxicité.

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Travail expérimental 1

Influence des solutions physiologiques sur la perméabilité

in vitro de molécules actives

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Travail expérimental 1

Nous avons montré dans la première partie du manuscrit le potentiel de la chambre de perméation d’Ussing dans les études de transport des molécules actives ou à partir d’une formulation à travers les tissus épithéliaux et monocouches de cellules polarisées.

Dans nos travaux expérimentaux, nous exploiterons le potentiel de cette technique in

vitro pour évaluer l’influence des milieux et solutions physiologiques utilisés dans les études de perméabilité membranaire ainsi que leurs influences sur la viabilité, l’intégrité et la perméabilité de la membrane épithéliale.

Dans cet objectif ; et pour déterminer l’influence que peuvent avoir les solutions sur la cinétique de passage membranaire in vitro, nous avons fait le choix d’évaluer la perméation de deux molécules hydrophiles, la vitamine C (VitC) et le paracétamol en chambre de perméation d’Ussing.

La vitamine C est choisie car son absorption intestinale est sodium-dépendante et selon les cas est passive pour des doses élevées et active pour des faibles doses Le paracétamol est choisi car son absorption intestinale est passive et paracellulaire Il serait donc intéressant d’observer le profil de passage intestinal de ces deux molécules dans des solutions de compositions ioniques différentes.

Enfin, la deuxième partie des travaux sera consacrée au resvératrol, molécule hydrophobe, avec une stratégie de formulation dont l’objectif principal est d’améliorer sa dissolution aqueuse et sa perméation intestinale en vue d’une meilleure biodisponibilité.

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Travail expérimental 1

L’influence des solutions physiologiques sur la biodisponibilité intestinale de médicaments in vitro.

L’objectif principal de ce travail était d’évaluer l’impact de la composition des différentes solutions physiologiques standards utilisées communément pour les études en chambre d’Ussing sur le passage des principes actifs et sur l’intégrité fonctionnelle de la membrane intestinale.

2. La chambre d’Ussing et passage des molécules actives

Les conditions d’utilisation des chambres de perméation d’Ussing et des équations qui régissent sont: 1) la température doit être constante et identique dans les deux compartiments (lumière intestinale comme côté séreux), 2) la pression qui s’exerce sur les deux côtés de la membrane doit être identique et constante, 3) le système doit être en état stationnaire des flux (J), c’est-à-dire que J doit être constant en fonction du temps, 4) pas d’interaction entre le flux de la molécule à mesurer et les autres constituants du système. Pour cette dernière condition est qui également la plus importante, pour avoir un système adéquate, il faut que l’activité spécifique de chacun des composants de la solution physiologique utilisé soit identique de part et d’autre de la membrane ainsi que le potentiel électrique des ions qui la compose.

Lorsqu’on modifie la quatrième condition d’utilisation des chambres d’Ussing, en modifiant la composition du milieu physiologique séreux et muqueux on constate une modification des paramètres électriques de la membrane, notamment le courant de court-circuit (Isc) de base et surtout la conductance (G), (voir figure1)

De plus, lorsque la membrane intestinale est placée en chambre d’Ussing pendant deux heures, on remarque une modification de la structure des villosités intestinales par rapport au témoin (Ringer seul). Le prétraitement du tissu avec une solution contenant du KCl à 2% provoque une altération des villosités intestinales.

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Intestinal drug absorption kinetics determined in vitro in Ussing

chambers is affected by the nature of the physiological media

Godefroy Mamadou 1,2, Bruno Eto2, Nicolas Limas-Nzouzi2, Nathalie Ialy-Radio3, Gilles Ponchel1

1Institut Galien Paris Sud, Faculté de Pharmacie, University of Paris 11, Châtenay- Malabry France

2 TransCell-Lab Laboratory, Faculty of Medicine Xavier Bichat, University of Paris Diderot - Paris7, Paris, France.

3Inserm U1149, Faculty of Medicine Xavier Bichat, University of Paris Diderot - Paris7, Paris, France.

ABSTRACT

After oral delivery, the intestinal absorption of the released drugs depends on many physico-chemical characteristics, including aqueous solubility, lipophilicity, the concentration of the drug dissolved in the intestinal medium, as well as the physiological characteristics of the mucosal membrane. Absorption kinetics through intestinal tissues can be very conveniently determined with Ussing chambers, enabling to simultaneously evaluate the tissue viability by the means of electrical parameters measurements and the determination of transepithelial fluxes (Jms) of the considered drug.

The objective of this in vitro study is to determine whether the nature of physiological medium affects the experimentally determined permeability values through the jejunum of rat. Acetaminophen and vitamin C were used because their absorption pathways are well characterized. Moreover, these substances are easily absorbed in vivo, making them ideal models for the present study.

The study showed that: (i) the composition of the physiologic solutions affected the conductance of mucosal intestinal membrane when they were added to the mucosal side only or to on both sides; (ii) the intestinal permeations of acetaminophen and vitamin C were affected by different media. After 2h, the trans intestinal fluxes of vitamin C were Jms = 31.4 ± 1.44 µg.hr-1.cm-2 with Ringer solution whereas with

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Sorensen solution it was: Jms= 51.1 ± 7.09 µg.hr-1.cm-2 and Jms = 15.8 ± 1.92 µg.hr1.cm -2 with Ringer Na+-free. The flux of acetaminophen was Jms = 17.9 ± 1.39 µg.hr1.cm-2

with Ringer solution whereas with Sorensen solution was Jms= 12.6 ± 1.99 µg.hr-1.cm -2 (iii) A correlation (r² = 0.979) was found between conductance and fluxes of vitamin C while using different solutions including Ringer, Sorensen, and Na+-free Ringer. With acetaminophen, we found a correlation only with Ringer solution (R² = 0.976).; (iv) The composition of the physiological solutions also affected the response of the tissue to different pharmacological effectors such as glucose and carbacholine; (v) This study confirmed that proximal jejunum, when incubated in Ussing chambers, exhibited less morphological deterioration of the villi of the epithelial cells, although the crypt structure remained intact.

In conclusion, this study showed that frequently used media could affect the functional viability of the intestinal tissue and could affect the experimental permeability data, suggesting that composition of physiological medium must be carefully taken into account during intestinal absorption studies of drugs in vitro.

Keywords: Intestinal absorption, physiological buffers composition, rat intestine, jejunum, physiological media, Ussing chambers, vitamin C, acetaminophen.

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1. INTRODUCTION

After oral delivery of solid dosage forms, the bioavailability of drugs depends on many physico-chemical characteristics of the drug, including aqueous solubility, drug ionizability, possible adsorption to mucus glycoproteins, variations in local dissolved drug concentrations along the gastrointestinal tract and simultaneously on the resistance of the mucosal intestinal membrane to the passage of the drug from the luminal side to the serosal side of the mucosa, this is often described by the apparent permeability coefficient of the drug. Although the main barrier against diffusion of many drugs is constituted by the tightly bound epithelial cells, the mucus gel layer which coats the luminal epithelial layer reinforces the barrier even further. As shown recently, it forms an unstirred aqueous layer which can significantly limit the intestinal permeability (Dossou-Yovo et al., 2014; Fairstein et al., 2013; Pappenheimer, 2001; Pelissier et al., 2010).In vitro, the condition of experimentation and tissue additional factors that might be taken into account in the evaluation of intestinal permeability of drugs. The transport of drugs, nutrients, xenobiotics and electrolytes can be different in jejunum, ileum or côlon (Swaan et al., 1994; Ungell et al., 1998).

From the experimental point of view, the intestinal permeability of the active ingredients can be estimated in humans by using different models: monolayers obtained through cell cultures, intestinal tissue put together in the Ussing chamber, the method of the turned around intestine, in vivo intestinal perfusion ... Among these techniques, Ussing chambers allow to study and evaluate realistic in vitro conditions, enough for the passage of molecules through the intestinal epithelium, from the physiological media. It also tracks the electrical parameters membrane characteristics and sustainability by monitoring certain physiological functions.

To the extent where many active substances require a suitable formulation due to their low aqueous solubility, instability or other factors, it would be very interesting to use the Ussing model, in order to study the effect of these formulations on the intestinal passage. This requires checking the sturdiness of the Ussing model in the conditions which are slightly diverted from the physiological conditions and of Ussing.

Indeed, in the Ussing model, the biological preparation (epithelial tissue, epithelial cells monolayer ...) are put together between two semi-chambers, defining a mucous compartment and a serosal compartment with the application conditions, temperature

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and maintenance pressure that needs to remain constant on either sides of the membrane and with the same ionic composition.

Therefore, experimentally speaking, it would be interesting to study the influence of the composition of some buffers which could be used for permeability and for studying the pharmaceutical formulations or the essential molecules and this way maintaining the electrical resistance of the tissue.

Theoretically, in permeability case studies with a complex physiological media might constitute the primary source for cellular toxicity, inducing a data error and leading to misinterpret the results.

Made up of one or more salts which can be added as additives such as albumin or ethanol ... these survival physiological solutions are an complete formulation, directly or indirectly influencing intestinal permeability or because they are in contact with the molecule model (instability of the molecule in the middle) or due to a toxic effect on the

Dans le document Evaluation de l'impact des formulations pharmaceutiques sur l'absorption intestinale des molécules actives au moyen des Chambres d’Ussing (Page 64-159)